Hormone composition

ABSTRACT

Twice weekly administration of an analog to a Vagifem tablet which only contains 10 μg of active material has a sufficient effect.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 10/016,858, filed Dec. 14, 2001. This patentapplication claims the benefit of U.S. Provisional Patent ApplicationNo. 60/260,182 filed Jan. 5, 2001, U.S. Provisional Patent Application60/260,183 filed Jan. 5, 2001, and U.S. Provisional Patent Application60/260,184 filed Jan. 5, 2001 and Danish Patent Application Nos. PA 200001890, filed Dec. 15, 2000, PA 2000 01891, filed Dec. 15, 2000, and PA2000 01892, filed Dec. 15, 2000.

The present invention relates to a composition containing oestrogen,which is to be administered vaginally.

BACKGROUND OF THIS INVENTION

Vaginal atrophy can occur in postmenopausal woman and estrogen deprivedwomen who actually do not need any systemic hormone replacement therapybut just local therapy. Consequently, local, topical treatment ispreferred in order to avoid the systemic side effects due tolong-lasting oestrogen therapy. Local therapy for this purpose has beenstudied for a long period of time and the hormone has been administeredas creams, gels, and silastic rings.

About every second postmenopausal women will experience urogenitaldiscomfort associated with estrogen deficiency. Previous studies haveshown that although many of these women use an oral hormone replacementtherapy, urogenital symptoms persist.

A composition commonly used is Vagifem® marketed by Novo Nordisk A/S.Vagifem is developed to treat estrogen deficiency-deprived atrophicvaginitis. Vagifem is a small tablet containing 25 μg 17β-estradiol. Forexample, reference can be made to Maturitas 14 (1991), 23-31, where thewomen initially received 25 μg estradiol for 2 weeks and, thereafter, 25μg estradiol once weekly or twice weekly. A usual treatment is onetablet of Vagifem containing 25 μg estradiol daily for the first 2 weeksof treatment and, thereafter, one tablet twice a week.

Conveniently, Vagifem is administered by placing a tablet at the top ofa slim-line pencil-like disposable applicator. By introducing theapplicator to the vagina, the Vagifem tablet will, due to the adhesivecharacteristics of Vagifem, stay in the vagina.

A pharmaceutical medicament for local, essentially non-systemic,treatment of vaginal dryness, in particular in the menopausal woman,characterized by a unit galenical formulation comprising a naturalestrogen selected from the group consisting of 17β-estradiol and itssalts and its derivatives in solution or in suspension in a lipophilicagent, with an estrogen content which corresponds to an equivalent unitdose of at most 15 μg, preferably less than 10 μg, of 17β-estradiol, ahydrophilic gel-forming bioadhesive agent, a gelling agent for thelipophilic agent, and a hydrodispersible agent, is described in U.S.Pat. No. 6,060,077. Hence, according to the 6,060,077 specification,17β-estradiol and its salts and its derivatives are in solution or insuspension. Consequently, it cannot be a tablet.

SUMMARY OF THIS INVENTION

One object of this invention is to furnish a hormone composition whichgives a clinical effect on vaginal symptoms which is as good as thatobtained by administration of Vagifem twice weekly.

A further object of this invention is to furnish a hormone compositionfurnishing no or only inferior systemic absorption.

A still further object of this invention is to furnish a hormonecomposition furnishing significant improvement in the vaginal mucosa.

A still further object of this invention is to furnish a hormonecomposition furnishing no or only inferior systemic effect.

A still further object of this invention is to furnish a hormonecomposition furnishing low absorption of estrogen.

A still further object of this invention is to furnish a hormonecomposition furnishing low serum concentration of estradiol.

A still further object of this invention is to furnish a hormonecomposition furnishing no or only inferior accumulation of circulatingestradiol.

A still further object of this invention is to furnish a hormonecomposition furnishing positive effects on an atrophic vaginalepithelum.

A still further object of this invention is to furnish a hormonecomposition furnishing complete or substantial vaginal maturation.

A still further object of this invention is to furnish a hormonecomposition furnishing a reduced risk of osteporosis.

A still further object of this invention is to furnish a hormonecomposition furnishing increases in percentage of superficial vaginalcells.

A still further object of this invention is to furnish a hormonecomposition which can be used for the treatment of atrophic vaginitis.

A still further object of this invention is to furnish a hormonecomposition furnishing a vaginal pH value below bout 5.5.

A very specific object of this invention is to furnish a hormonecomposition furnishing all or most of the following characteristics:Relief of vaginal symptoms, improved urogenital atrophy, decreasedvaginal pH, and improved cytologic maturation of both the vaginal andurethral mucosa.

DETAILED DESCRIPTION OF THIS INVENTION

The vaginal symptoms treated by the use according to the presentinvention are dryness, soreness, irritation, and dyspareunia. Theurogenital health is characterized by secretions, epithelial integrity,surface thickness, and the pH value of the vagina.

Surprisingly, it has been found that the use according to the claimsbelow have pharmaceutical and clinical advantages compared with theknown uses of similar compositions.

It is often recommended to precede the use according to the claims belowwith a treatment with a somewhat higher dosage of an estrogen, forexample, estradiol. Such a treatment is herein designated apre-treatment. In a preferred embodiment, this pre-treatment is thedaily treatment with the same dose as that used for a bi-weekly useaccording to the claims below.

The present invention relates to use of an oestrogen in the manufactureof a composition containing oestrogen for the treatment of atrophicvaginitis in woman, by administering weekly an amount of about 10 toabout 30 μg estradiol to a woman. According to a preferred embodiment,this invention relates to the use wherein the women treated ismenopausal or post-menopausal women. According to a further preferredembodiment, this invention relates to the use wherein weekly an amountof about 15 to about 25 μg estradiol is administered. According to afurther preferred embodiment, this invention relates to the use whereindaily about 1.5 to about 4 μg estradiol is administered. According to afurther preferred embodiment, this invention relates to the use whereindaily about 2 to about 3 μg estradiol is administered. According to afurther preferred embodiment, this invention relates to the use whereintwice weekly about 5 to about 15 μg estradiol is administered. Accordingto a further preferred embodiment, this invention relates to the usewherein twice weekly about 7 to about 13 μg estradiol is administered.According to a further preferred embodiment, this invention relates tothe use wherein twice weekly about 9 to about 11 μg estradiol isadministered. According to a further preferred embodiment, thisinvention relates to the use wherein no progestogen is administered.According to a further preferred embodiment, this invention relates tothe use wherein the composition is to be administered vaginally.According to a further preferred embodiment, this invention relates tothe use wherein it is used for a period of time of more than 2 weeks,preferably more than 1 month, more preferred more than 2 months, andeven more preferred more than 3 months. According to a further preferredembodiment, this invention relates to the use wherein administration isperformed using a tablet. Furthermore, the present invention relates toa method of treating atrophic vaginitis, comprising administering acomposition as described in any of the previous use claims.

The compositions used according to this invention may be preparedanalogously to the preparation of similar compositions, for example,Vagifem. The compositions used according to this invention may containany constituent used or suggest to be used in similar compositions. Thecompositions used according to this invention may be administeredanalogously with the administration of similar compositions. All theseaspects are known to the skilled art worker.

According to a preferred embodiment of this invention, the compositiondealt with herein is a tablet. According to a preferred embodiment, thecomposition dealt with herein consists of about 60% through about 80%hypromellose (or another convenient matrix former), about 20% throughabout 25% lactose (or another convenient filler), about 5% through about15% maize starch (or another convenient filler), about 0.2% throughabout 1.5% magnesium stearate (or another convenient lubricant) andabout 0.2% through about 5% film-coating, as well as E2. In a morepreferred embodiment, the composition consists of about 65% throughabout 70% hypromellose (or another convenient matrix former), about 20%through about 24% lactose (or another convenient filler), about 8%through about 12% maize starch (or another convenient filler), about0.3% through about 1.3% magnesium stearate (or another convenientlubricant) and about 0.3% through about 3% film-coating, as well as E2.In an even more preferred embodiment, the composition consists of about67% hypromellose, about 22% lactose, about 10% maize starch, about 0.5%magnesium stearate and about 1% film-coating. According to a preferredembodiment of this invention, each tablet contains, in addition to theactive material, about 53.7 mg hypromellose, about 17.9 mg lactosemonohydrate, about 8 mg maize starch, about 0.4 mg magnesium stearateand the film-coating consisting of about 0.5 mg hypromellose and about0.06 mg macrogel 6000 (polyethylene glycol 6000 NF).

On a dry basis. In the final tablets, the content of water is preferablybelow 10%, more preferred below 7%. All percentages ratios given hereare per weight basis.

One way of preparing the tablets is via the following steps: Suspensionof estradiol, granulation, blending, compression, preparation offilm-coating solution, and film-coating.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, in any combination thereof, be material forrealizing the invention in diverse forms thereof. Especially,interesting and surprising effects are dealt with and described inexamples 2 & 3.

EXAMPLE 1

58 postmenopausal women were treated with tablets containing either 10or 25 μg 17β-estradiol. The women inserted 1 tablet intravaginally, oncedaily for the initial 2 weeks of the study and then twice per week(Sunday & Thursday) for the following 10 weeks. Hence, some of the womenonly received tablets containing 10 μg 17β-estradiol and the remainingwomen only received tablets containing 25 μg 17β-estradiol. Theestradiol profile when administering 25 or 10 μg 17β-estradiol wassimilar after the first dose (zero weeks of treatment) and after theabove continuous treatment with 25 or 10 μg 17β-estradiol twice weeklyfor 10 weeks.

EXAMPLE 2

Treatment of atrophic vaginitis according to the present invention withlow-dose 17β-estradiol tablets results in consistent, low absorption ofestradiol without accumulation.

Objectives:

The vaginal absorption of 17β-estradiol (hereinafter designated E2) wasevaluated and two low doses E2 (25 μg and 10 μg) were compared inpostmenopausal women with atrophic vaginitis.

Design:

In a double-blind, randomized, parallel-group study, 58 postmenopausalwomen were treated with either 25 or 10 μg E2 for 12 weeks. Serum E2 andfollicle stimulating hormone (hereinafter designated FSH) concentrationswere measured throughout the study at specified intervals. The areaunder the curve, maximal concentration, and time to maximalconcentration were determined for serum E2 concentrations. Maturationvalues of vaginal mucosal cells were assessed as an indicator of changesin the condition of the vaginal mucosa in response to treatment.

Results:

For both treatment groups, the E2 profiles were similar at weeks 0 and12. The mean E2 concentrations, areas under the curve, and maximalconcentrations were higher in the 25-μg E2 group than in the 10-μg E2group. For the majority of patients in each treatment group, the areasunder the curve remained below 600 μg·hr/mL at each time point, and themean FSH concentrations were in the normal postmenopausal range.Patients in each treatment group showed significant improvement (P≦0.01)in the condition of the vaginal mucosa.

Conclusion:

Treatment with either 25- or 10-μg E2 vaginal tablets resulted inimprovements in the vaginal mucosa and low absorption of estrogenwithout the systemic effects often associated with ERT. After 12 weeksof therapy for atrophic vaginitis, absorption patterns remainedconsistent, and patients did not experience an accumulation ofcirculating E2.

Studies have shown that vaginal ERT preparations can result in rapid andefficient absorption of E2 into systemic circulation. However, low-dosepreparations that contain 10 and 25 μg E2 effectively relieve thesymptoms of atrophic vaginitis without unwanted systemic side effects. Alow-dose (25 μg) E2 vaginal tablet (Vagifem®; Novo Nordisk, Denmark) hasbeen developed to treat estrogen deficiency-derived atrophic vaginitis.These vaginal tablets contain a film-coated hydrophilic cellulose matrixthat adheres well to the vaginal mucosa and hydrates slowly to provide acontrolled release of E2. They are designed to provide estrogenizationof the vaginal mucosa while preventing significant increases in serumestrogen concentrations. In this study, the vaginal absorption of E2 wasevaluated and two low doses of E2 (25 μg and 10 μg) were compared inpostmenopausal women with atrophic vaginitis.

MATERIALS AND METHODS

This single-center, randomized, double-blind, parallel-group study wasconducted in Atlanta, Ga. The study was approved by the appropriateinstitutional review board, and written informed consent was obtainedfrom each patient. The study was conducted in compliance with theDeclaration of Helsinki of 1975, revised in 1983.

In this study, generally healthy, postmenopausal women (hysterectomizedor nonhysterectomized), aged 45 years or older, were enrolled. Patientshad no more than 5% superficial cells, as assessed by vaginal cytologyevaluation, and serum E2 concentrations no greater than 20 pg/mL.Nonhysterectomized patients had endometrial thicknesses no greater than4 mm, as determined by pelvic ultrasound. Patients with known orsuspected history of breast cancer or other hormone-dependent tumors,acute thrombophlebitis or thromboembolic disorders associated withprevious estrogen use, or vaginal infection requiring further treatment(at baseline) were excluded from the study, as were patients withgenital bleeding of unknown etiology (within 12 months prior toscreening). Patients were not to have used any type of vaginal, oral, orvulvar preparations within 7 days prior to screening; any exogenouscorticosteroid or sex hormones within 8 weeks prior to baseline; anyinvestigational new drug within the past 30 days; or diethylstilbestrol.

After the screening visit, patients received no study treatment duringthe 4-week run-in period prior to the baseline visit. At the baselinevisit, patients were randomized to receive vaginal tablets containingeither 25 or 10 μg E2 on a 1:1 basis using a computer-generated scheme.The vaginal tablets were identical in appearance. Patients inserted 1tablet intravaginally, once daily for the initial 2 weeks of the studyand then twice per week (Sunday and Thursday) for the remaining 10weeks. Patients were instructed to use their medication at a consistenttime each day, preferably in the morning. After the baseline visit,patients returned to the clinic at weeks 1, 2, 4, 8, and 12 formeasurements of serum E2 and FSH, as well as assessments of vaginalcytology.

Upon presentation to the clinic for each visit, a vaginal cytologyspecimen was obtained. Patients then inserted the tablets. Blood sampleswere drawn 30 minutes before tablet insertion, and at 1, 2, 4, 5, 6, 7,8, 10, 12, and 24 hours after insertion to determine the serum E2concentration via radioimmunoassay. The blood samples obtained at 30minutes before insertion, and at 6, 12, and 24 hours after insertionwere also used to determine the FSH concentration via immunoradiometricassay.

The maturation value of vaginal mucosal cells was calculated from thepercentages of parabasal, intermediate, and superficial cells accordingto the following equation:maturation value=0×[parabasal cells, %]+0.5×[intermediate cells,%]+1.0×[superficial cells, %]

The pharmacokinetic parameters of area under the concentration-timecurve from 30 minutes before tablet insertion to 24 hours after tabletinsertion, maximal concentration, and time to maximal concentration weredetermined for serum concentrations of E2. Data for area under the curveand maximal concentration were converted to a logarithmic scale, andchanges from first dose (at the baseline visit) in the logarithmicvalues were estimated using 95% confidence intervals derived from pairedt-tests. Differences between treatment groups in the degree ofabsorption of E2 were determined using 95% confidence intervals derivedfrom two sample t-tests based on the observed mean values of thelogarithms of area under the curve and maximal concentration.Differences within treatment groups in FSH concentration were determinedusing the Wilcoxon signed rank test based on changes from baseline inmean concentrations at weeks 2 and 12. Mean concentrations were definedas the average concentrations obtained at 30 minutes before tabletinsertion and 6, 12, and 24 hours after insertion. Baselineconcentrations for E2 and FSH were defined as the values observed at 30minutes before tablet insertion at the baseline visit.

This manuscript presents data for the evaluable patient population,which was defined as those patients who had serum E2 concentrationsbelow 20 μg/mL at baseline and who had complete data available at thebaseline visit and weeks 2 and 12.

RESULTS

A total of 58 women were treated with vaginal tablets containing either25 μg E2 (28 women) or 10 μg E2 (30 women). Ten women discontinuedprematurely from the study. The evaluable patient population consistedof 42 women; 19 women received 25 μg E2, and 23 women received 10 μg E2.Demographic and baseline characteristics for the evaluable patientpopulation are presented in Table 1. Patient characteristics weresimilar between treatment groups, with the exception of percentage ofparabasal cells at baseline, which was significantly lower for patientsin the 25-μg E2 group compared to those in the 10-μg E2 group (P=0.027,t-test).

The 24-hour concentration profiles for serum E2 at weeks 0 and 12 arepresented in FIGS. 1 and 2, respectively, and the associatedpharmacokinetic characteristics are presented in Table 2. At weeks 0 and12, the serum E2 profiles were similar within each treatment group. Theserum E2 concentrations, as well as the corresponding mean area underthe curve and maximal concentration, were higher for patients whoreceived 25 μg E2 than for patients who received 10 μg E2. The averageserum E2 concentrations over 24 hours were also higher in the 25-μg E2group than in the 10-μg E2 group.

A comparison between the areas under the curve for serum E2 at weeks 0and 12 is presented in FIG. 3. The majority of patients in eachtreatment group had areas under the curve below 600 pg·hr/mL at bothtime points (14 patients [74%] and 22 patients [96%] in the 25- and10-μg E2 groups, respectively). A comparison between area under thecurve for serum E2 and mean FSH concentration at week 12 is presented inFIG. 4. At week 12, the majority of patients in each treatment group hadmean FSH concentrations in the normal postmenopausal range (at least 35pg/mL); 3 patients in the 25-μg E2 group had mean FSH concentrationsbelow 35 pg/mL.

The mean maturation value and mean change from baseline in maturationvalue are presented in Table 3. In each treatment group, patientsexperienced a significant increase in maturation value over baselinevalues (P≦0.001 at weeks 1 and 2, and P≦0.01 at week 12; 2-tailed,paired t-test). At all time points, mean maturation values and meanchanges from baseline in maturation value were comparable betweentreatment groups. A comparison between the area under the curve forserum E2 and the maturation value at week 12 is presented in FIG. 5. Themajority of patients in each treatment group (13 patients [68%] and 14patients [64%] in the 25- and 10-μg E2 groups, respectively) showedincreases in maturation values from the corresponding baseline values(53.4 and 51.0 in the 25- and 10-μg E2 groups, respectively).

CONCLUSIONS

The optimum intravaginal therapy will provide consistent estrogenabsorption with adequate relief of vaginal symptoms without systemicabsorption and the associated side effects. The low-dose vaginal tabletsused in this study meet these criteria.

This study examined the systemic absorption of E2 in patients whoreceived treatment with either 25- or 10-μg E2 vaginal tablets for 12weeks. The majority of patients in each treatment group (74% in the25-μg E2 group and 96% in the 10-μg E2 group) experienced low systemicabsorption of E2 at both the beginning and end of the 12-week treatmentperiod, as indicated by areas under the serum E2 concentration curvebelow 600 pg·hr/mL at each time point. Of the 6 remaining patients, 4who did experience higher E2 absorption at week 12 also had areas underthe curve greater than 600 pg·hr/mL at both week 0 and 12, suggestingthat these patients were characteristically high E2 absorbers. It islikely that these patients would experience greater absorption of E2 asa result of any ERT.

The 24-hour serum E2 profiles at weeks 0 and 12 were similar for eachtreatment group, again indicating that overall, women had consistent E2absorption patterns at the beginning and end of the treatment period.The average E2 concentrations at each time point were within the normalpostmenopausal range (normal postmenopausal range for E2 concentration:≦40 pg/mL). The promising results from this study demonstratedconsistent E2 absorption over 12 weeks of treatment.

In this study, after 12 weeks of treatment with either 25 or 10 μg E2,FSH concentrations were rarely suppressed to premenopausal levels,suggesting that the observed increase in serum E2 concentration is notassociated with clinically significant systemic E2 potency. Both the 25-and 10-μg E2 dose levels demonstrated positive effects on an atrophicvaginal epithelium while maintaining low serum concentrations of E2. Theimprovement in vaginal health may be due to direct perfusion and/orlymphatic absorption of the local E2 through the vaginal epithelium. Inthis study, vaginal maturity was measured exclusively with thematuration value. Since the vaginal response is likely due to enhancedglycogenization and acidification of the vagina, monitoring the vaginalpH would provide another useful measure of vaginal health. Vaginalmaturation with low concentrations of circulating E2 is a primarytreatment goal of local vaginal ERT. Reduced risks of osteoporosis inpostmenopausal women have also been observed. These benefits likely relyon the concentration of circulating E2 added to the endogenousproduction of E2 in bone, which is especially true in older, naturalpostmenopausal women. Since the average serum E2 concentrations werehigher for patients who recieved 25 μg E2 than for those who received 10μg E2, it is possible that patients who receive the lower dose mayderive additional benefits because of very low likelihood of anysystemic effect. TABLE 1 Demographic and Baseline Characteristics(Evaluable Patients) Treatment group 25 μg E2 10 μg E2 Characteristic (N= 19) (N = 23) Age (yr)^(a) 52.1 ± 5.6 (45-63) 54.8 ± 5.1 (48-69) RaceCaucasian 16 (84.2%) 18 (78.3%) Other 3 (15.8%) 5 (21.7%) Time sincelast 10.7 ± 7.6 (1-25) 14.3 ± 8.7 (1-32) menses (yr)^(a) HysterectomizedYes 12 (63.2%) 17 (73.9%) No 7 (36.8%) 6 (26.1%) E2 concentration at 7.0± 2.8 (3-13) 7.6 ± 3.7 (2-18) screening (pg/mL)^(a) Vaginal cytology atscreening Parabasal cells (%)^(a) 1.9 ± 2.5^(b) (0-7) 8.4 ± 12.9^(b)(0-48) Intermediate cells (%)^(a) 95.2 ± 7.8 (65-100) 90.1 ± 12.4(51-100) Superficial cells (%)^(a) 2.9 ± 8.0 (0-35) 1.5 ± 1.7 (0-6)SD = standard deviation;E2 = estradiol^(a)Data presented as mean ± SD (range).^(b)Statistically significant; P = .027 (t-test)

TABLE 2 Pharmacokinetic Parameters for 24-Hour Serum Estradiol Profiles(Evaluable Patients) Treatment group Pharmacokinetic 25 μg E2 10 μg E2Time point characteristic (N = 19) (N = 23) Week 0 Area under the curve538 ± 265 349 ± 107 (pg · hr/mL)^(a) Maximal concentration 51 ± 34 35 ±17 (pg/mL)^(a) Time to maximal 15 ± 9  9 ± 5 concentration (hr)^(a)Serum concentration 22 15 over 24 hours (pg/mL) Week 12 Area under thecurve 563 ± 341 264 ± 120 (pg · hr/mL)^(a) Maximal concentration 49 ± 2722 ± 17 (pg/mL)^(a) Time to maximal 13 ± 6  10 ± 8  concentration(hr)^(a) Serum concentration 23 11 over 24 hours (pg/mL)E2 = estradiol;SD = standard deviation^(a)Data presented as mean ± SD.

TABLE 3 Mean Maturation Values and Changes From Baseline (All Patients)Time Treatment group point N 25 μg E2 N 10 μg E2 Week 0 Mean ± SD 2552.4 ± 7.1 28 51.0 ± 6.2  Week 12 Mean ± SD 20 58.4 ± 7.5 23 62.2 ± 15.7Mean change ± SD 20   7.0 ± 8.7^(b) 23  11.2 ± 17.8^(b)SD = standard deviation^(a)Statistically significant; P ≦ .001 (2-tailed, paired t-test)^(b)Statistically significant; P ≦ .01 (2-tailed, paired t-test)

EXAMPLE 3

Treatment according to the present invention with low-dose 17β-estradioltablets relieves vaginal symptoms, improves urogenital atrophy (vaginalhealth), and increases maturation of the vaginal and urethral epithelia(mucosa) without abnormal endometrial growth.

Objectives:

Vaginal tablets containing 25 or 10 μg 17β-estradiol (herein designatedE2) or placebo were evaluated and compared in postmenopausal women withatrophic vaginitis.

Methods:

In a multicenter, randomized, double-blind, placebo-controlled,parallel-group study, 230 post-menopausal women received treatment with25 or 10 μg E2 or placebo for 12 weeks. Efficacy was measured withcomposite scores of vaginal symptoms (dryness, soreness, and irritation)and vaginal health (secretions, epithelial integrity, surface thickness,and pH). Vaginal and urethral cytology analyses were also performed, andthe vaginal maturation value was determined. Safety assessments includedendometrial biopsies.

Results:

Greater improvements in composite scores for vaginal symptoms andvaginal health characteristics were reported for patients in the activetreatment groups than in the placebo group. Significantly greaterimprovements were reported at Weeks 7 and 12 (P≦0.05). At Week 12, over75% of patients in the active treatment groups had vaginal pH valuesbelow 5.5 compared to approximately 40% of patients in the placebogroup. Both vaginal and urethral cytology analyses indicated largerincreases in percentages of superficial cells in the active treatmentgroups than in the placebo group. Correspondingly, increases in vaginalmaturation value were higher in the active treatment groups than in theplacebo group. One patient who received 25 μg E2 had an abnormal biopsyresult.

Conclusions:

Both the 25- and 10-μg E2 vaginal tablets provided relief of vaginalsymptoms, improved vaginal health, and increased maturation of both thevaginal and urethral mucosa without abnormal endometrial growth.

INTRODUCTION

As endogenous estrogen production declines during menopause, the vaginaand other estrogen-dependent tissues gradually undergo atrophic changes.The loss of estrogen-influenced cellular maturation results in acondition identified as atrophic vaginitis. The symptoms of atrophicvaginitis include dryness, soreness, irritation, and dyspareunia.Additionally, the vaginal epithelium becomes more susceptible toinfection and secondary inflammation. Oral estrogen therapy has beenassociated with metabolic side effects as well as breast and endometrialhyperplasia.

Novo Nordisk has developed Vagifem™, a low-dose estrogen vaginal tabletthat contains 25 μg E2 in a hydrophilic cellulose-based matrix.Pharmacokinetic studies of Vagifem™ have shown that in an atrophicvaginal epithelium, vaginally administered E2 is readily absorbed, butafter normalization and maturation of the epithelium, E2 absorption issignificantly reduced.

This study evaluated and compared the efficacy and safety of vaginaltablets containing 25 or 10 μg E2 or placebo during 12 weeks of therapyfor vaginal atrophy.

METHODS AND MATERIALS

This phase III, multicenter, randomized, double-blind,placebo-controlled, parallel-group study was conducted at 9 centers inthe United States. The study was approved by the appropriateinstitutional review boards, and informed consent was obtained from eachpatient prior to beginning study procedures. The study was conducted incompliance with the Declaration of Helsinki of 1975, revised in 1983.

Women at least 45 years of age or older with moderate-to-severe vaginaldryness and soreness were enrolled. All patients were required to haveserum E2 concentrations of 20 pg/mL or less and to have no more than 5%superficial vaginal cells. Patients with intact uteri were also requiredto be at least 12 months past natural menopause with an endometrialthickness of 5 mm or less.

Patients with creatinine levels greater than 1.4 mg/dL, bilirubin levelsgreater than 1.2 mg/dL, aspartate transaminase levels greater than 50U/L, or hemoglobin levels less than 11.5 g/dL were excluded from thestudy. Patients with a known or suspected history of breast carcinoma,hormone-dependent tumor, genital bleeding of unknown etiology, acutethrombophlebitis or thromboembolic disorder associated with estrogenuse, vaginal infection requiring treatment, allergy to the test drug orits constituents, or any serious disease or chronic condition that couldinterfere with study compliance were also excluded from the study. Theuse of an investigational drug within the 30 days preceding screening,any homeopathic preparation within the 7 days preceding study druginitiation, any exogenous corticosteroid or sex hormones within the 8weeks preceding study drug initiation, or diethylstilbestrol wasprohibited.

The purpose of this study was to compare 25 and 10 μg E2 and placebo.Using a computer-generated randomization scheme, subjects (patients)were randomized using a 2:2:1 ratio to receive vaginal tablets thatcontained 25 μg E2, 10 μg E2, or placebo. All vaginal tablets wereidentical in appearance. Patients inserted 1 tablet daily for 14 days.Thereafter, patients inserted 1 tablet twice per week (Sunday andThursday) for the remainder of the trial. Patients were to insert thetablets at the same time each day (preferably at bedtime). Patients wereevaluated for efficacy and safety at Week—4 (screening), Week 0(baseline), and Weeks 2, 7, and 12.

Efficacy assessments included patient ratings of atrophic vaginitissymptoms, investigator ratings of vaginal health, and vaginal andurethral cytology. Patients used intensity ratings of none, mild,moderate, or severe to evaluate atrophic vaginitis symptoms (dryness,soreness, irritation, dyspareunia, and vaginal discharge). Intensityratings were assigned ascending scores from 0 (none) to 3 (severe) foranalysis. A composite score for atrophic vaginitis symptoms was definedas the average of the individual symptom scores for dryness, soreness,and irritation. This composite score did not include scores fordyspareunia (which was not evaluated by all patients) or vaginaldischarge (which was rated as none or mild by the majority of patients).The composite score and the change from baseline for the composite scorewere examined at each time point. Differences within and betweentreatment groups were analyzed using an analysis of variance (ANOVA).

Investigators used a severity scale of none, mild, moderate, or severeto assess vaginal health characteristics (secretions, epithelialintegrity, surface thickness, color, and pH). Severity categories wereassigned ascending scores from 0 (none) to 3 (severe) for analysis. Toavoid multiple endpoint issues, composite scores were defined. Acomposite score for vaginal health was defined as the average of theindividual vaginal health characteristic scores. The composite score andthe change from baseline for the composite score were examined at eachtime point. Differences within and between treatment groups wereanalyzed using an analysis of variance (ANOVA).

Vaginal and urethral cell samples were harvested and analyzed byindependent cytologists to determine the percentages of parabasal,intermediate, and superficial cells. Maturation values were calculatedaccording to the following equation:maturation value=1.0×[superficial cells, %]+0.5×[intermediate cells, %]Endometrial biopsies were performed at the end of the study in patientswith intact uteri. The number of patients with abnormal biopsies wascompared between treatment groups.

RESULTS

A total of 91 women received 25 μg E2, 92 women received 10 μg E2, and47 women received placebo. Demographic and baseline characteristics didnot differ significantly between treatment groups, with the exception ofrace (Table 4). The percentage of nonwhite patients was significantlylower in the 25-μg E2 group than in the placebo group (P=0.026,Cochran-Mantel-Haenszel test). Nine patients (9.9%) in the 25-μg E2group, 18 patients (19.6%) in the 10-μg E2 group, and 8 patients (17.0%)in the placebo group discontinued prematurely from the study.

The vaginal symptom composite score profiles between Weeks 0 and 12 arepresented in FIG. 6. At Week 0, the vaginal symptom composite scoresmeasured approximately 1.9 in each treatment group. At Weeks 2, 7, and12, vaginal symptom composite scores were significantly lower than thecorresponding baseline values for each treatment group (P≦0.001;two-tailed paired t-test). In the active treatment groups (the 25- and10-μg E2 groups), vaginal symptom composite scores continued to decreaseafter Week 0 and measured approximately 0.5 and 0.6 at Week 12,respectively. In contrast, in the placebo group, vaginal symptom scoresremained nearly constant after Week 0 and measured approximately 1.1. AtWeeks 7 and 12, the differences from baseline observed in the activetreatment groups were significantly larger than those observed in theplacebo group (P≦0.01 and P≦0.05 in the 25- and 10-μg E2 groups,respectively; two-tailed linear model analysis).

The urogenital (vaginal) health composite score profiles between Weeks 0and 12 are presented in FIG. 7. At Week 0, the vaginal health compositescores measured approximately 1.7 in each treatment group. At Weeks 2,7, and 12, vaginal health composite scores were significantly lower thanthe corresponding baseline values for each treatment group (P≦0.01;two-tailed paired t-test). At Weeks 2, 7, and 12, the decreases invaginal health composite scores observed in the active treatment groupswere significantly larger than those observed in the placebo group(P≦0.001; two-tailed linear model analysis). At Week 7, the decrease invaginal health composite score was significantly larger in the 25-μg E2group than in the 10-μg E2 (P=0.004, two-tailed linear model analysis).

The number and percentage of patients with vaginal pH values below 5.5at Weeks 0, 2, 7, and 12 are presented in Table 5. At Week 0,approximately 35% of patients in each treatment group had vaginal pHvalues below 5.5. At Weeks 2, 7, and 12, the percentage of patients withvaginal pH values below 5.5 increased from the baseline percentages foreach treatment group. These increases were significantly greater forpatients in the active treatment groups than in the placebo group(P≦0.05; two-tailed linear model analysis). At Week 12, over 75% ofpatients in the active treatment groups and approximately 40% ofpatients in the placebo group had vaginal pH values below 5.5.

The percentage of superficial cells from vaginal cytology analysis atWeeks 0, 2, 7, and 12 are presented in FIG. 8. At all time points afterWeek 0, subjects in the active treatment groups showed eithersignificant increases (P≦0.05) or trends toward increases in thepercentage of superficial cells compared with subjects in the placebogroup. These increases are presented in Table 7.

The maturation values at Weeks 0 through 12 are presented in FIG. 9. AtWeek 0, the maturation values measured approximately 45% in eachtreatment group. At each time point, the maturation values weresignificantly higher than the corresponding baseline values for eachtreatment group (P≦0.01; two-tailed paired t-test). The increases frombaseline values were larger in the active treatment groups than in theplacebo group. At Week 12, the maturation values measured approximately60% in the active treatment groups and approximately 55% in the placebogroup. At Weeks 2 and 7, the increases in maturation values observed inthe 25-μg E2 group were significantly larger than those observed in theplacebo group (P≦0.05; two-tailed linear model analysis). At Week 2, theincrease in maturation value observed in the 10-μg E2 group wassignificantly larger than that observed in the placebo group (P=0.001;two-tailed linear model analysis).

The percentage superficial cells from urethral cytology analysis atWeeks 0, 2, 7, and 12 are presented in FIG. 10. At all time points afterWeek 0, subjects in the active treatment groups showed eithersignificant increases (P≦0.05) or trends toward increases in thepercentage of superficial cells compared with subjects in the placebogroup. These increases are presented in Table 8.

The percentage of superficial vaginal and urethral cells at Weeks 0 and12 are presented in FIGS. 11(a), (b), and (c) for the 25- and 10-μg E2groups and the placebo group, respectively. At Week 0, the majority ofthe subjects in each treatment group had percentages of both superficialvaginal and urethral cells less that or equal to 5% (57 subjects [81%],53 subjects [85%], and 34 subjects [97%] in the 25- and 10-μg E2 groupsand the placebo group, respectively). At Week 12, more subjects in theactive treatment groups that in the placebo group had increases inpercentages of both superficial vaginal and urethral cells (52 subjects[74%], 44 subjects [71%], and 21 subjects [60%] in the 25- and 10-μg E2groups and the placebo group, respectively).

The endometrial biopsy results at Week 12 are presented in Table 6. Ofsubjects with biopsies that yielded sufficient tissue, 1 subject in the25-μg E2 group showed simple hyperplacia without atypia. However, therewas no pretreatment biopsy for comparison.

The percentages of parabasal, intermediate, and superficial cells fromvaginal cytology analysis at Weeks 0, 2, 7, and 12 are presented in FIG.12. In most cases, the percentages of cells in each category changedsignificantly from the corresponding baseline values (P≦0.01; two-tailedpaired t-test). At each time point, the percentage of superficial cellsincreased. At Weeks 2 and 7, the increases in percentage of superficialcells observed in the 25-μg E2 group were significantly larger thanthose observed in the placebo group (P≦0.003; two-tailed linear modelanalysis). At Weeks 2 and 12, the differences in percentage ofsuperficial cells observed in the 10-μg E2 group were significantlylarger than those observed in the placebo group (P≦0.035; two-tailedlinear model analysis).

The percentages of parabasal, intermediate, and superficial cells fromurethral cytology analysis at Weeks 0, 2, 7, and 12 are presented inFIG. 13. In most cases, the percentages of superficial and parabasalcells changed significantly from the corresponding baseline values(P≦0.05; two-tailed linear model analysis). Generally, the percentage ofsuperficial cells increased, and the percentages of intermediate andparabasal cells decreased. At Weeks 2 and 7, the increases in percentageof superficial cells observed in the 25-μg E2 group were significantlylarger than those observed in the placebo group (P≦0.044; two-tailedlinear model analysis). At Week 2, the increases in percentage ofsuperficial cells observed in the 10-μg E2 group were significantlylarger than those observed in the placebo group (P≦0.018; two-tailedlinear model analysis).

CONCLUSIONS

In this 12-week study, treatment with either 25- or 10-μg E2 tabletsresulted in greater improvement in vaginal symptoms (as assessed by thepatients) and vaginal health (as assessed by the investigators) thantreatment with placebo. At each time point after baseline, improvementsin the urogenital (vaginal) health composite scores were significantlygreater in the active (25-μg and 10-μg E2) group than in the placebogroup (P≦0.01). At each time point after 2 weeks of treatment,improvements in the vaginal symptom composite scores were alsosignificantly greater (P≦0.05). Additionally subjects in the activetreatment groups had statistically significant increases (P≦0.05) ortrends toward increases in the percentage of superficial vaginal cellscompared with subjects in the placebo group. In this study, treatmentwith 25- or 10-μg E2 resulted in comparable improvements as assessed byboth patients and investigators.

Improvements in the symptoms of atrophic vaginitis become physicallyevident and manifest as changes in the vaginal mucosa. The condition ofthe vaginal mucosa can be determined through vaginal cytologymeasurements and maturation value. In this study, the percentages ofimmature parabasal and intermediate cells decreased, consequentlyincreasing the percentages of more mature superficial cells in eachtreatment group. These changes are reflected in significant increasesover baseline values in maturation value in each treatment group(P≦5.01). After 12 weeks of treatment, the maturation values forpatients in the 25- or 10-μg E2 groups were approximately 60%, while thematuration values for patients in the placebo group were approximately55%.

A second clinical measure of vaginal activity is the vaginal pH, acomponent of the urogenital (vaginal) health composite score. Asestrogen production declines after menopause, lactobacilli, whichproduce lactic acid from vaginal glycogen, disappear from the vaginalflora and vaginal pH increases. Consequently, higher vaginal pH valuesare associated with a lack of estrogen in the vaginal mucosa. In thisstudy approximately twice as many patients who received 25 or 10 μg E2than those who received placebo had vaginal pH values below 5.5 after 12weeks of treatment (75% versus 40%, respectively). The results fromanalysis of vaginal cytology and pH indicate a positive effect of the25- and 10-μg E2 vaginal tablets on estrogenation of the vaginalepithelum (mucosa).

The lower portions of the vaginal and urinary tracts have the sameembryological origin, and genital tract disorders such as atrophicvaginitis are often accompanied by atrophic changes in the urinary tractthat may include dysuria, stress incontinence, and urinary tractinfections. Consequently, estrogen therapy may also have an effect onthe urethral epithelium. In this study, the condition of the urethralepithelium was determined through urethral cytology. Similar to thevaginal cytology analysis, the percentages of parabasal cells in theurethral epithelium decreased and the percentage of superficial cellsincreased for each treatment group. Although this study was not designedto further determine benefits to the urinary tract, this urethralmaturation could be attributed to estrogenation of the urethral mucosa.

Thus, although the 25-μg E2 vaginal tablets used in this study appear topositively affect the vaginal and urethral epithelia, they were notassociated with endometrial abnormalities. TABLE 4 Demographic andBaseline Characteristics Treatment group 25 μg E2 10 μg E2 PlaceboCharacteristic (N = 91) (N = 92) (N = 47) Age (yr)^(a) 58.3 ± 7.4(46-78) 57.7 ± 6.5 (46-79) 57.6 ± 4.8 (50-70) Race White 88 (96.7%) 83(90.2%) 41 (87.2%) Nonwhite 3 (3.3%)^(b) 9 (9.8%) 6 (12.8%)^(b) Timesince last 14.8 ± 9.6 (1-40) 13.5 ± 7.8 (1-34) 13.6 ± 8.1 (1-33) menses(yr)^(a) Hysterectomized Yes 42 (46.2%) 44 (47.8%) 23 (48.9%) No 49(53.8%) 48 (52.2%) 24 (51.1%)SD = standard deviation^(a)Mean ± SD (range)^(b)Statistically significant; P = .026 (Cochran-Mantel-Haenszel test)

TABLE 5 Number and Percentage of Patients With Vaginal pH Values Below5.5 Treatment group 25 μg E2 10 μg E2 Placebo Time point n/N (%) n/N (%)n/N (%) Week 0 31/90 (34.4) 27/89 (30.3) 17/46 (37.0) Week 2 64/87(73.6)^(a) 67/84 (79.8)^(b) 21/43 (48.8) Week 7 71/83 (85.5)^(b,d) 57/80(71.3)^(c) 23/44 (52.3) Week 12 63/79 (79.7)^(b) 54/71 (76.1)^(b) 15/38(39.5)A two-tailed linear model analysis was used to compare treatment groupsat each time point.^(a)Comparison with placebo, statistically significant; P ≦ .01^(b)Comparison with placebo, statistically significant; P ≦ .001^(c)Comparison with placebo, statistically significant; P ≦ .05^(d)Comparison with 10 μg 17 β-E2, statistically significant; P ≦ .05

TABLE 6 Endometrial Biopsy Results at Week 12 Treatment group 25 μg E210 μg E2 Placebo Result (N = 32) (N = 32) (N = 21) Normal^(a) 28 (87.5%)25 (78.1%) 18 (85.7%) Abnormal^(b) 1 (3.1%) 0 (0.0%) 0 (0.0%) Other^(c)3 (9.4%) 7 (21.9%) 3 (14.3%)^(a)Results indicative of an atrophic endometrium, weakly proliferativeendometrium, proliferative endometrium, or secretory endometrium wereclassified as normal.^(b)Results indicative of endometrial hyperplasia (simplex, complex, oratypical) or carcinoma were classified as abnormal.^(c)Results indicative of a menstrual endometrium, mucosal polyps,insufficient tissue, or other finding were classified as other.

TABLE 7 Mean and mean change from baseline in percentage of superficialvaginal cells Treatment Group Time 25 μg E2 10 μg E2 Placebo Point NMean Change N Mean Change N Mean Change Week 86 4.0 79 3.1 45 4.3 0 Week85 34.2 30.7^(a) 76 28.3 25.0^(a) 42 13.1 8.6 2 Week 80 28.2 23.9^(b,c)72 20.4 17.1 41 15.1 10.4 7 Week 75 19.9 15.4 68 20.1 16.9^(d) 36 13.89.0 12A two-tailed linear model analysis was used to compare treatment groupsat each time point.^(a)Comparison with placebo, statistically significant; P ≦ .001^(b)Comparison with placebo, statistically significant; P ≦ .01^(c)Comparison with 10 μg E2, statistically significant; P ≦ .05^(d)Comparison with placebo, statistically significant; P ≦ .05

TABLE 8 Mean and mean change from baseline in percentage of superficialurethral cells Treatment Group Time 25 μg E2 10 μg E2 Placebo Point NMean Change N Mean Change N Mean Change Week 86 3.2 83 2.5 42 3.0 0 Week83 21.9 19.2^(a,b) 79 16.7 14.1^(c) 34 6.5 3.3 2 Week 77 17.6 14.2^(d)70 13.5 10.7 38 8.6 5.5 7 Week 70 11.2  7.5 64 9.5  6.8 34 7.0 3.6 12A two-tailed linear model analysis was used to compare treatment groupsat each time point.^(a)Comparison with placebo, statistically significant; P ≦ .001^(b)Comparison with 10 μg E2, statistically significant; P ≦ .05^(c)Comparison with placebo, statistically significant; P ≦ .01^(d)Comparison with placebo, statistically significant; P ≦ .05

EXAMPLE 4

Manufacture of 10 μg Tablets

Suspension of Estradiol

10.3 g estradiol hemihydrate (equivalent to 10.0 g estradiol, anhydrous)is suspended in a solution of hypromellose (14.8 g) in purified water15.6 L) while stirring.

Granulation

Blending, granulation, and drying are performed in a fluid bed.

Hypromellose (53.69 kg), lactose monohydrate (17.90 kg), and maizestarch (8.00 kg) are sucked into the fluid bed through a sieve and thenblended.

The granulation takes place by spraying the suspension of estradiol onthe mixture of excipients. After spraying the granulate is dried.

Blending

Sieving is performed and magnesium stearate (400 g) is blended into thegranulate

Compression

The tablets are compressed using a rotary tablet press.

Preparation of Film-Coating Solution

Hypromellose (400 g) and macrogol 6000 (50 g) are dissolved in purifiedwater (9.55 kg)

Film-Coating

In a coating pan, the tablets are coated with the coating solution(0.576 mg dried substance/tablet), using an air atomizing spray system.After coating sampling of tablets to release testing takes place.

EXPLANATION TO THE FIGURES

FIG. 1. Serum concentrations of estradiol at week 0.

25 μg E2

10 μg E2 FIG. 2. Serum concentrations of estradiol at week 12.

25 μg E2

10 μg E2 FIG. 3. Area under the serum estradiol curve at weeks 0 and 12.

25 μg E2

10 μg E2 FIG. 4. Area under the serum estradiol curve and serum FSHconcentration at week 12.

25 μg E2

10 μg E2 FIG. 5. Area under the serum estradiol curve and maturationvalue at week 12. Maturation values at baseline were 52.4 in the 25-μgE2 group and 51.0 in the 10-μg E2 group.

25 μg E2

10 μg E2 FIG. 6. Vaginal symptom composite score profiles - subjects whoreceived at least 1 dose of study medication, had baseline assessment,and had at least 1 post-baseline assessment

Placebo

E2 to 10 μg

E2 to 25 μg FIG. 7. Vaginal health composite score profiles - subjectswho received at least 1 dose of study medication, had baselineassessment, and had at least 1 post-baseline assessment

Placebo

E2 to 10 μg

E2 to 25 μg FIG. 8. Vaginal cytology results (percentage of superficialcells) - subjects who received at least 1 dose of study medication, hadbaseline assessment, and had at least 1 post-baseline assessment.

10 μg E2

Superficial

25 μg E2 FIG. 9. Maturation values - subjects who received at least 1dose of study medication, had baseline assessment, and had at least 1post-baseline assessment

Placebo

E2 to 10 μg

E2 to 25 μg FIG. 10. Urethral cytology results - subjects who receivedat least 1 dose of study medication, had baseline assessment, and had atleast 1 post-baseline assessment

10 μg E2

Placebo

25 μg E2 FIG. 11. Percentages of superficial vaginal and urethralcells - subjects who had superficial vaginal and urethral cellassessments at Weeks 0 and 12 (a) 25 μg E2 (b) 10 μg E2 (c) Placebo FIG.12. Vaginal cytology results ν Parabasal

Intermediate

Superficial (a) Placebo (b) 10 μg E2 (c) 25 μg E2 FIG. 13. Urethralcytology results ν Parabasal

Intermediate

Superficial (d) Placebo (e) 10 μg E2 (0 25 μg E2

1. A method for treating atrophic vaginitis in a patient in need of suchtreatment, said method comprising administering vaginally to saidpatient an amount of about 10 μg estradiol, wherein administration ofsaid amount occurs once or twice per week.
 2. A method according toclaim 1, wherein the patient is a menopausal or post-menopausal woman.3. A method according to claim 1, wherein no progestogen isadministered.
 4. A method according to claim 1, wherein said at leastonce-weekly administration occurs over a time period of more than twoweeks.
 5. A method according to claim 4, wherein said period of time ismore than 3 months.
 6. A method according to claim 1, wherein saidestradiol is administered in tablet form and wherein each tabletcomprises, in addition to estradiol or a therapeutically equivalentamount of a salt thereof, hypromellose, lactose monohydrate, maizestarch, and magnesium stearate.
 7. A method according to claim 6,wherein said tablet comprises about 60-80% w/w hypromellose; 20-25% w/wlactose; about 5-15% maize starch; and about 0.2-1.5% magnesiumstearate.
 8. A method for treating atrophic vaginitis in a patient inneed of such treatment, said method comprising administering vaginallyto said patient a tablet comprising hypromellose, lactose monohydrate,maize starch, and magnesium stearate.
 9. A method according to claim 8,wherein said tablet comprises about 60-80% w/w hypromellose; 20-25% w/wlactose; about 5-15% maize starch; and about 0.2-1.5% magnesiumstearate.